Vascular endothelial growth factor (VEGF), acidic fibroblast growth factor (aFGF), platelet-derived growth factor (PDGF), and insulin-like growth factor-1 (IGF-1) are active modulators of cell proliferation, with profound effects on tumor cell growth, and, in the case of PDGF, intimal smooth muscle cell growth. In addition, certain such growth factors stimulate blood vessel formation, or angiogenesis. Inhibition of angiogenesis is an important therapeutic goal in the control of tumor development in cancer patients.
Vascular disease treatment by balloon angioplasty, implantation of a stent, or by resection (e.g. a vascular graft), results in local damage to the endothelial cell lining of the vessel wall. A result of the damage is that the smooth muscle cells of the intima begin to proliferate in a PDGF-dependent process termed intimal hyperplasia or restenosis. Despite this side-effect, percutaneous transluminal coronary angioplasty, stent insertion, and vascular by-pass and grafting remain primary treatments for many patients with coronary artery disease and can relieve myocardial ischemia by reducing lumen obstruction and improving coronary flow. A very significant proportion of patients develop restenosis within three months. Restenosis results in significant morbidity or frequently necessitates further interventions such as repeat angioplasty or coronary bypass surgery.
Platelet-derived growth factor (PDGF) is a potent inducer of growth and motility in several cell types such as fibroblasts, endothelial cells and smooth muscle cells. It induces cell proliferation, angiogenesis, wound healing, chemotaxis and inhibits apoptosis. PDGF has also been directly implicated in malignant diseases involving uncontrolled cell proliferation such as cancer where overexpression of PDGF and/or PDGF receptors is common in human tumors including glioblastomas and sarcomas. In addition to its role in stimulating uncontrolled growth of cancer cells, PDGF also stimulates the proliferation and migration of endothelial cells leading to formation of new blood vessels, a process that is required for the growth of tumors. Furthermore, PDGF has also been shown to stimulate endothelial cells to express high levels of vascular endothelial growth factor (VEGF) a potent angiogenesis inducer. Finally, the importance of PDGF in angiogenesis has also been documented by the fact that mice deficient in PDGF-BB or its receptor β are defective in blood vessel development.
PDGF elicits the above biological response by binding to its cell surface receptor, PDGFR, a tyrosine kinase. Binding results in dimerization, receptor auto (cross) phosphorylation and recruitment via the resulting phosphotyrosine of SH2 domain-containing signaling proteins such as Grb2/SOS1, PLC-γ, PI-3kinase and Src. These proteins trigger several arms of the PDGF signal transduction pathways that are involved in different cellular responses. For example, Grb2/Sos1 activates the GTPase Ras which in turn results in the activation of a cascade of mitogen-activated protein kinases (MAPK), such as Erk1 and Erk2, and this contributes to the proliferation arm of the PDGF signaling pathway. Others such as PI3-kinase activate yet another kinase, AKT, which is responsible for the survival or the anti-apoptotic arm of PDGF signaling pathways.
The involvement of PDGF overactivity in several diseases with excessive proliferation prompted many researchers to target PDGF as a therapeutic strategy. The approaches that have been used to interfere with aberrant PDGF function in disease include PDGFR antibodies that block PDGF binding, linear or cyclic peptides corresponding to areas of PDGF that bind its receptor, receptor dimerization antagonists, and inhibitors of the tyrosine kinase activity of the receptor. An area that has been underexploited in the search of antagonists of PDGF overactivity is that of rationally designed agents that bind to PDGF and prevent it from activating its receptor. In this area only a few strategies such as antibodies against PDGF, soluble forms of PDGFR and DNA aptamers have been tried and those have had only marginal success. This is primarily due to the difficulty of designing agents to disrupt protein-protein interactions that are mediated over large surface binding areas, and because such molecules are often swiftly degraded by enzymes.
An attractive approach to producing synthetic molecules capable of binding to defined biological targets, involves synthetic antibody mimics disclosed by Hamilton and Yoshitomo in U.S. Pat. No. 5,770,380, issued Jun. 23, 1998, and which is hereby incoporated in its entirety by reference. Such molecules have the advantages of biological stability and can be rationally designed and screened. However, no such antibody mimics have previously been produced that specifically bind growth factors.
Here we describe a novel approach that targets growth factor surfaces, such as the regions of PDGF (loops I and III) that are involved in binding to its receptor, to overcome the aforementioned limitations in the art. These advantages and more will be apparent to those of skill in the art upon reading the following disclosure.